The discovery and the extensive biochemical and biophysical characterization of the evolutionarily conserved cytoplasmic protein modular domains (such as SH2, SH3, PH, and WW domains) have revolutionized the way protein-protein interactions in cellular signal transduction is understood [Pawson and Scott, Nature 278: 2075-2080 (1997)]. Since these protein module-mediated interactions play an important role in regulating numerous cellular processes including cell growth, proliferation, differentiation and apoptosis, studies of the protein domain-mediated pathways have revealed the molecular basis of various human diseases. This has led to the discovery of new drug targets for treatment of these diseases.
The Phosphorylated Tyrosine Binding (PTB) domain (also called PID or SAIN domains) is another evolutionarily conserved cytoplasmic protein modular domain involved in cellular signal transduction. The PTB domain was first identified as a protein module that could bind phosphorylated tyrosines, and thus was classified as an alternative to the Src homology 2 (SH2) domain [Blaikie et al. J. Biol. Chem. 269:32031-32034 (1994); Kavanaugh, and Williams, Science 266:862-1865 (1994); O'Neill et al., Mol. Cell. Biol. 14: 6433-6442 (1994)]. However, PTB domains are structurally and functionally distinct from SH2 domains, and recognize amino acid residues amino-terminal (rather than carboxyl-terminal) to the phosphotyrosine (pY) [Zhou and Fesik, S. W. Prog. Biophys. Molec. Biol. 64:221-235 (1995)]. In particular, PTB domains preferentially bind to phosphorylated proteins at sites containing an NPXpY motif and hydrophobic amino acids amino-terminal to this sequence [Gustafson et al. Mol. Cell. Biol. 15:2500-2508 (1995); Kavanaugh et al. Science 268:1177-1179 (1995); Zhou et al., J. Biol. Chem. 270:31119-31123 (1995); Trub et al., J. Biol. Chem. 270:18205-18208 (1995); Singyang, J. Biol. Chem. 270:14863-14866 (1995)]. Moreover, unlike SH2 domains, PTB domains typically show very low protein sequence homology. Different PTB domains exhibit distinct selectivity for residues amino-terminal to the NPXpY motif (SEQ ID NO:6). For example, the PTB domain of the insulin receptor substrate 1. (IRS-1) favors hydrophobic residues at different structural locations of its receptor, than those preferred by the PTB domain of the adaptor protein Shc. Recent studies demonstrate that the PTB domains of X11 and Numb can also recognize sequences related to the NPXpY motif in tyrosine-phosphorylation and non-phosphorylation-dependent manners [Li et al. Proc. Natl. Acad. Sci. USA 94: 7204-7209 (1997); Li et al. Nat., Struct. Biol. 5:1075-1083 (1998); and Zhang et al., EMBO J. 16:6141-6150 (1997)].
The NMR structural analyses of the Shc and IRS-1 PTB domains revealed the detailed structural basis of their protein recognition [Zhou et al., Nature 378:584-592 (1995); and Zhou et al, Nature Struct. Biol. 3:388-393 (1996)]. Despite their very low sequence homology, the PTB domains of Shc and IRS-1 consist of a conserved pleckstrin (PH) domain fold, i.e. a β-sandwich containing two nearly orthogonal, anti-parallel β-sheets capped at one end by an amphipathic C-terminal α-helix. Furthermore as stated above, the structurally related PTB domains of Shc and IRS-1 employ two very different mechanisms for recognizing the phosphotyrosine and the hydrophobic residues amino-terminal to the NPXpY sequence. For example, for Shc, an Ile residue of a synthetic peptide derived from TRKA receptor (HIIENPQpYFSDA, SEQ ID NO:7) binds in a deep hydrophobic pocket located between P5 and the C-terminal α-helix. The corresponding residue of a peptide derived from interleukin-4 receptor (IL-4R) (LVIAGNPApYRS, SEQ ID NO:4), binds on the surface of the protein. In addition, the IRS-1 PTB domain recognizes Ile and Leu of the IL-4R peptide through interactions with a hydrophobic site on the surface of a second β-sheet, whereas the analogous site in Shc is not available for peptide binding, because it is covered by a loop and the N-terminal portion of an α-helix. Notably, in contrast to SH2 domains, key arginines that are important for binding to phosphotyrosine are located in different regions of different PTB domain sequences. SNT (suc1-associated neurotrophic factor target protein) proteins are two newly discovered insulin receptor substrate-like (IRS-like) signaling adaptor molecules that are specifically activated by receptors for fibroblast growth factors (FGF), nerve growth factors (NGF), and glial-derived neurotrophic factor, but are not activated by most other growth factor receptor kinases [Xu et al., J. Biol. Chem. 273:17987-17990 (1998); Kouhara et al. Cell 89:693-702 (1997); Meakin et al., J. Biol. Chem. 274:9861-9870 (1999)]. SNT tyrosine phosphorylation promotes activation of Ras/MAPK and SHP-2, two biochemical pathways critical for ligand-induced biological responses. Current studies suggest that SNT activation enables FGFs and NGFs to elicit specific biological responses not achieved by activation of other receptor tyrosine kinases, which fail to stimulate SNTs. It has further been shown that the activation and tyrosine-phosphorylation of SNTs require direct contact between receptors and the amino-terminal phosphotyrosine binding domain of each SNT [Xu et al., J. Biol. Chem. 273:17987-17990 (1998); Meakin et al., J. Biol. Chem. 274:9861-9870 (1999)]. Whereas SNT PTB domains recognize a canonical NPXpY motif on NGF receptors such as TRKA, they unexpectedly bind a tyrosine- and asparagine-free motif in the juxtamembrane segment of the FGF receptor (FGFR). Thus, the SNT PTB domain represents a very unique protein modular domain, which can specifically bind two seemingly unrelated receptor peptide moieties.
The structural and functional diversity of PTB domains is further demonstrated in the SNT PTB domains. Sequence homology alignment and secondary prediction analysis using an approach of profile-based neural network predictions [PHD method; EMBL-Heidelberg et al., Mol. Bol. 232, 584-599 (1993); Rost and Sander, Proteins 19:55-77 (1994)] reveals that SNT PTB domains contain a large insert sequence (predicted to be an α-helix) located between the corresponding strand P7 and the C-terminal α-helix in the Shc and the IRS-1 PTB domains. It is interesting to note that whereas in all of the published three-dimensional structures of PTB and PH domains (regardless of whether they were determined by NMR or X-ray crystallography) structural variations have been found in different loop regions, none of have been observed between the P7 and the C-terminal α-helix region.
One means of modulating cellular proliferation and/or differentiation is to either inhibit or facilitate the interaction of the PTB domain of an SNT and the FGF receptor. Therefore, there is a need to identify agonists or antagonists to the SNT/FGFR complex. Unfortunately, such identification has heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure based drug design. In this case, the three dimensional structure of SNT/FGFR complex is determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995); and Dunbrack et al., Folding & Design, 2:27-42 (1997)]. However, heretofore the three-dimensional structure of the SNT/FGFR complex has remained unknown. Therefore, there is a need for obtaining a form of the SNT/FGFR complex that is amenable for NMR analysis and/or X-ray crystallographic analysis. Furthermore there is a need for the determination of the three-dimensional structure of such complexes. Finally, there is a need for procedures for related structural based drug design predicated on such structural data.
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